A number of laboratories, including those involved in this Program Project, have demonstrated that Adeno-associated virus holds significant promise for the correction of human disease. These experiments have shown that AAV can be used to transfer genes into primary cells both in vivo and ex vivo. However, if the development of AAV as a vector is to move forward several issues must be addressed about the basic biology of AAV. First, the AAV terminal repeats, which are absolutely required for DNA replication, integration and packaging, have not been completely characterized. The terminal repeats, which are absolutely required for DNA replication, integration and packaging, have not been completely characterized. The terminal repeats (TRs) consist of several elements that we and others have identified, including, the Rep binding element (RBE), the CTTTG motif within the B and C palindromes, the terminal resolution site (trs) and the secondary structure of the TR. Although a substantial amount of information has accumulated about the RBE, no one has yet determined the precise sequences within the trs or the CTTTG motif that are essential for DNA replication, nor has anyone investigated the spatial and orientation requirements of the elements within the TR for Rep functions. Additionally, no one has clearly demonstrated which multimeric Rep complex is functional for trs nicking or DNA helicase activity. Second, although it is clear that both AAV DNA replication and latency require cellular replication and/or repair enzymes, nothing is known about the enzymes that are involved. We recently developed an in vitro AAV DNA replication assay which faithfully recapitulates the mechanism of AAV DNA replication. We propose to use this assay to identify and characterize the cellular enzymes required for AAV DNA replication using standard biochemical fractionation and reconstitution techniques. Our goal is to reconstitute AAV DNA replication in vitro with purified enzymes. Finally, relatively little is known about the functional regions of the AAV capsid or the mechanism of encapsidation. For this reason we will isolate and characterize mutations within the AAV capsid proteins to identify functional regions within the capsid gene required for packaging. These mutations will be useful for designing rAAV vectors that are capable for targeting specific cell types. We will also take advantage of our recently developed in vitro packaging reaction to study the mechanism of AAV encapsidation. Our specific aims are: 1: Characterization of the AAV origin sequences and their interaction with Rep protein. 2: Purification and identification of cellular DNA replication enzymes required for AAV DNA replication in vitro. 3: To study AAV DNA packaging and capsid structure.